Please wait a minute...
浙江大学学报（医学版）
浙江大学学报（医学版）  2018, Vol. 47 Issue (1): 35-40    DOI: 10.3785/j.issn.1008-9292.2018.02.05
专题报道     
MyD88非依赖性信号通路在枸杞多糖抑制糖尿病小鼠肿瘤坏死因子α中的作用
刘婷婷(),王凌霄,杨晓辉,姚智卿,蔡慧珍*()
宁夏医科大学公共卫生与管理学院, 宁夏 银川 750004
TLR/NF-κB independent signaling pathway in TNF-α suppression of diabetic MyD88-knockout mice after Lycium barbarum polysaccharides administration
LIU Tingting(),WANG Lingxiao,YANG Xiaohui,YAO Zhiqing,CAI Huizhen*()
School of Public Health and Management, Ningxia Medical University, Yinchuan 750004, China
 全文: PDF(1031 KB)   HTML( 13 )
摘要：

目的: 研究枸杞多糖对髓样分化因子88（MyD88）非依赖性信号通路的影响，探讨枸杞多糖影响TNF-α生成的机制。方法: 采用高糖高脂饲料联合链脲菌素诱导MyD88基因敲除小鼠形成2型糖尿病模型。将造模成功的小鼠随机分为模型对照组、阳性对照组和枸杞多糖组，另取8只小鼠作为健康对照组。干预三个月后，采用实时定量RT-PCR和蛋白质印迹法检测小鼠腹腔巨噬细胞中TRAM、TRIF、TRAF6、RIP1和TNF-α基因和蛋白表达，ELISA法测定小鼠血清TNF-α水平。结果: 枸杞多糖抑制了糖尿病小鼠腹腔巨噬细胞中Tram、Trif、Traf6和Tnf-α的基因表达，激活了Rip1基因（均P < 0.05），但对TRAM、TRIF、TRAF6、RIP1和TNF-α蛋白表达和血清TNF-α水平无影响（均P > 0.05）。结论: 枸杞多糖可能不能通过MyD88非依赖性信号通路抑制TNF-α的生成。
关键词： 髓样分化因子88/生理学;  信号传导;  肿瘤坏死因子α/生物合成;  多糖类/药理学;  枸杞/化学;  糖尿病, 2型;  疾病模型, 动物;  细胞, 培养的    
Abstract:

Objective: To investigate the effect of Lycium barbarum polysaccharides (LBPs) on TLR/NF-κB independent pathway and serum tumor necrosis factor (TNF-α) level in diabetic MyD88-knockout mice. Methods: Diabetes was induced by feeding high-fat/high-sugar diet and injection of low-dose streptozotocin in MyD88-knockout mice. The diabetic mice were randomly divided into model group, positive control group and LBPs group. The expressions of TRAM, TRIF, TRAF6, RIP1 and TNF-α mRNA and proteins in mouse peritoneal macrophages were detected by real-time RT-PCR and Western blotting after LBPs treatment for 3 month. Serum TNF-α was determined with ELISA kit. Results: Real time RT-PCR showed that compared with model group, the relative expressions of Tram, Trif, Traf6 and Tnf-α mRNA in macrophages of LBPs group were significantly decreased and expression of Rip1 was significantly increased (all P < 0.05). Expression of TRAM, TRIF, TRAF6, RIP1 and TNF-α proteins as well as serum TNF-α level had no significant difference between LBPs group and model group (all P > 0.05). Conclusion: LBPs may not inhibit serum TNF-α level through TLR/NF-κB independent pathway.
Key words: Myeloid differentiation factor 88/physiology    Signal transduction    Tumor necrosis factor-alpha/biosynthesis    Polysaccharides/pharmacology    Lycium barbarum/chemistry    Diabetes mellitus, type 2    Disease models, animal    Cells, cultured
收稿日期: 2017-12-03 出版日期: 2018-06-12
CLC:  R96  
基金资助: 国家自然科学基金(81460494);宁夏高等学校科学研究项目(NGY2013072)
通讯作者: 蔡慧珍     E-mail: 18695272282@163.com;xingcao_c@sina.com
作者简介: 刘婷婷(1993-), 女, 硕士研究生, 主要从事营养与慢性病研究; E-mail:18695272282@163.com; https://orcid.org/0000-0003-3176-9317
服务　　
把本文推荐给朋友
加入引用管理器
E-mail Alert
RSS
作者相关文章　　
刘婷婷
王凌霄
杨晓辉
姚智卿
蔡慧珍

引用本文:

刘婷婷,王凌霄,杨晓辉,姚智卿,蔡慧珍. MyD88非依赖性信号通路在枸杞多糖抑制糖尿病小鼠肿瘤坏死因子α中的作用[J]. 浙江大学学报（医学版）, 2018, 47(1): 35-40.

LIU Tingting,WANG Lingxiao,YANG Xiaohui,YAO Zhiqing,CAI Huizhen. TLR/NF-κB independent signaling pathway in TNF-α suppression of diabetic MyD88-knockout mice after Lycium barbarum polysaccharides administration. J Zhejiang Univ (Med Sci), 2018, 47(1): 35-40.

链接本文:

http://www.zjujournals.com/med/CN/10.3785/j.issn.1008-9292.2018.02.05    或    http://www.zjujournals.com/med/CN/Y2018/V47/I1/35

基因名称 引物序列(5′→3′) 片段大小
(bp)
Tram 正向：GTTTGCTCAGTGCGAGAGGA
反向：ATTCAGTTAGCTGGGAAGTGGT		 70
Trif 正向：CACGATCCTGCTCCTGACTG
反向：CTGTGGAGCAGTCTGGTTGT		 174
Rip1 正向：CTACCGGGTGTCAGGAATCA
反向：CAAGCCTCTTCAAACCGGGG		 183
Traf6 正向：TCATTATGATCTGGACTGCCCAAC
反向：TTATGAACAGCCTGGGCCAAC		 150
Tnf-α 正向：AAGAGGCACTCCCCCAAAAG
反向：GTGGTTTGTGAGTGTGAGGGT		 209
Actin 正向：CATCCGTAAAGACCTCTATGCCAAC
反向：ATGGAGCCACCGATCCACA		 171
表 1  实时定量RT-PCR引物序列
($\bar x \pm s$)
组别 Trif Tram Traf6 Rip1 Tnf-α
  *与健康对照组比较，P < 0.05；#与模型对照组比较，P < 0.05；△与阳性对照组比较，P < 0.05.Trif：β干扰素Toll/IL-1R结构域衔接蛋白；Tram：TRIF相关接头分子；Traf6：肿瘤坏死因子受体相关因子6；Rip1：受体相互作用蛋白1.
健康对照组 1.02±0.30 0.99±0.04 1.00±0.03 1.00±0.09 1.00±0.05
模型对照组 22.69±6.35* 1.84±0.16* 2.16±0.04* 12.97±2.77* 11.18±0.79*
阳性对照组 1.21±0.40# 0.42±0.03*# 0.81±0.39# 10.57±1.09* 7.38±2.21*#
枸杞多糖组 3.01±0.38# 0.24±0.10*#△ 0.66±0.19# 21.56±0.84*#△ 4.83±0.31*#△
表 2  各组腹腔巨噬细胞中Trif、Tram、Traf6、Rip1和Tnf-α mRNA表达量比较
图 1  各组腹腔巨噬细胞中TRIF、TRAM、TRAF6、RIP1和TNF-α蛋白表达电泳图
($\bar x \pm s$)
组别 TRIF TRAM TRAF6 RIP1 TNF-α
   *与健康对照组比较，P < 0.05；#与模型对照组比较，P < 0.05；△与阳性对照组比较，P < 0.05.TRIF：β干扰素Toll/IL-1R结构域衔接蛋白；TRAM：TRIF相关接头分子；TRAF6：肿瘤坏死因子受体相关因子6；RIP1：受体相互作用蛋白1.
健康对照组 0.93±0.25 1.04±0.17 0.33±0.26 1.23±0.21 0.21±0.08
模型对照组 0.82±0.24 1.00±0.27 0.37±0.19 1.09±0.14 0.46±0.11*
阳性对照组 0.94±0.24 1.41±0.53 0.34±0.11 1.33±0.16# 0.27±0.11#
枸杞多糖组 0.90±0.15 0.94±0.28 0.32±0.07 1.03±0.19△ 0.34±0.03
表 3  各组腹腔巨噬细胞中TRIF、TRAM、TRAF6、RIP1和TNF-α蛋白表达量比较
1 ZHAO R , JIN R , CHEN Y et al. Hypoglycemic and hypolipidemic effects of lycium barbarum polysaccharide in diabetic rats[J]. CHM, 2015, 7 (4): 310- 315
2 SHI G J , ZHENG J , WU J et al. Beneficial effects of Lycium barbarum polysaccharide on spermatogenesis by improving antioxidant activity and inhibiting apoptosis in streptozotocin-induced diabetic male mice[J]. Food Funct, 2017, 8 (3): 1215- 1226
doi: 10.1039/C6FO01575A
3 WU M, GUO L. Anti-fatigue and anti-hypoxic effects of lycium barbarum polysaccharides[C]//Proceedings of the International Conference on Advances in Energy, Environment and Chemical Engineering. Paris: Atlantis Press, 2015: 686-689.
4 CAI H , LIU F , ZUO P et al. Practical application of antidiabetic efficacy of lycium barbarum polysaccharide in patients with type 2 diabetes[J]. Med Chem, 2015, 11 (4): 383- 390
doi: 10.2174/1573406410666141110153858
5 MAY M J , GHOSH S . Signal transduction through NF-kappa B[J]. Immunol Today, 1998, 19 (2): 80- 88
doi: 10.1016/S0167-5699(97)01197-3
6 BRASIER A R . The NF-kappaB regulatory network[J]. Cardiovasc Toxicol, 2006, 6 (2): 111- 130
doi: 10.1385/CT:6:2
7 AKIRA S , TAKEDA K . Toll-like receptor signalling[J]. Nat Rev Immunol, 2004, 4 (7): 499- 511
doi: 10.1038/nri1391
8 解鸿翔, 周红 . 信号分子TRIF的研究进展[J]. 细胞与分子免疫学杂志, 2012, 28 (2): 217- 220
XIE Hongxiang , ZHOU Hong . Research progress on signal molecule TRIF[J]. Chinese Journal of Cellular and Molecular Immunology, 2012, 28 (2): 217- 220
9 YAMAMOTO M , SATO S , HEMMI H et al. TRAM is specifically involved in the Toll-like receptor 4-mediated MyD88-independent signaling pathway[J]. Nat Immunol, 2003, 4 (11): 1144- 1150
doi: 10.1038/ni986
10 OFENGEIM D , YUAN J . Regulation of RIP1 kinase signalling at the crossroads of inflammation and cell death[J]. Nat Rev Mol Cell Biol, 2013, 14 (11): 727- 736
doi: 10.1038/nrm3683
11 超晨. β干扰素TIR结构域衔接蛋白(TRIF)在NOD小鼠1型糖尿病发病中的作用[D]. 长沙: 中南大学, 2013.
CHAO Chen. The role of TIR-domain-containing adapter-inducing interferon-β(TRIF) in the development of type 1 diabetes in nonobese diabetic mice[D]. Changsha: Central South University, 2013. (in Chinese)
12 LAN P , LI W , WEN T N et al. iTRAQ protein profile analysis of arabidopsis roots reveals new aspects critical for iron homeostasis[J]. Plant Physiol, 2011, 155 (2): 821- 834
doi: 10.1104/pp.110.169508
13 VALENCIA-SANCHEZ M A , LIU J , HANNON G J et al. Control of translation and mRNA degradation by miRNAs and siRNAs[J]. Genes Dev, 2006, 20 (5): 515- 524
doi: 10.1101/gad.1399806
14 徐海波, 闫晓光, 钟威 . 新诊断2型糖尿病患者血清Nesfatin-1、肿瘤坏死因子-α水平与胰岛素抵抗的相关性研究[J]. 中国糖尿病杂志, 2017, 25 (1): 45- 48
XU Haibo , YAN Xiaoguang , ZHONG Wei . Correlation analysis of serum Nesfatin-1, TNF-α and insulin resistance in patients with newly diagnosed type 2 diabetes[J]. Chinese Journal of Diabetes, 2017, 25 (1): 45- 48
15 曹艳丽, 谷剑秋, 王涤非 et al. 肥胖者脂肪组织中TNF-α表达与脂肪细胞大小的相关性分析[J]. 中国组织化学与细胞化学杂志, 2013, 22 (4): 282- 285
CAO Yanli , GU Jianqiu , WANG Difei et al. Correlation between TNF-α expression in adipose tissue and adipocyte size in obesity[J]. Chinese Journal of Histochemistry and Cytochemistry, 2013, 22 (4): 282- 285
16 向宇飞. 糖尿病人群及高脂饮食肥胖小鼠的免疫代谢学研究[D]. 长沙: 中南大学, 2011.
XIANG Yufei. The immunometabolism studies in diabetic subjects and diet induced obese mice[D]. Changsha: Central South University, 2011. (in Chinese)
[1] 潘宗富, 方琦璐, 张轶雯, 李莉, 黄萍. 基于生物信息学的未分化甲状腺癌关键发病机制及其潜在干预靶点研究[J]. 浙江大学学报（医学版）, 2018, 47(2): 187-193.
[2] 丁京京,卢韵碧. 受体相互作用蛋白家族在炎症中的作用研究进展[J]. 浙江大学学报（医学版）, 2018, 47(1): 89-96.
[3] 王晓蓉,卢韵碧,张纬萍,魏尔清,方三华. 半胱氨酰白三烯受体对小鼠小胶质细胞吞噬功能的调节作用[J]. 浙江大学学报（医学版）, 2018, 47(1): 10-18.
[4] 魏振龙,石文贵,陈克明,周建,王鸣刚. 淫羊藿素通过CXCR4/SDF-1信号通路促进小鼠成骨细胞成熟和矿化[J]. 浙江大学学报（医学版）, 2017, 46(6): 571-577.
[5] 张斌斌 等. 抑制哺乳动物雷帕霉素靶蛋白信号通路对慢性脑缺血小鼠认知功能的改善和机制[J]. 浙江大学学报（医学版）, 2017, 46(4): 405-412.
[6] 封盛 等. 糖皮质激素受体信号通路在膀胱癌治疗中的作用研究进展[J]. 浙江大学学报（医学版）, 2016, 45(6): 655-660.
[7] 韩艳霞, 尤良顺, 刘辉, 毛莉萍, 叶琇锦, 钱文斌. 细胞周期蛋白依赖激酶抑制剂诱导HL-60细胞凋亡及分子机制研究[J]. 浙江大学学报（医学版）, 2015, 44(2): 174-178.
[8] 张春阳, 祝艳, 冯华松, 陈旭昕. 放射线照射的肺成纤维细胞对人脐带间充质干细胞中经典Wnt/β-catenin通路的影响[J]. 浙江大学学报（医学版）, 2015, 44(2): 162-166.
[9] 郑永霞，张成文，惠斌，等. 磷脂酰肌醇3激酶调节亚基基因对HepG2细胞增殖的影响[J]. 浙江大学学报（医学版）, 2014, 43(5): 559-.
[10] 石文贵，马小妮，陈克明. 初级纤毛在细胞信号转导中的作用与机制[J]. 浙江大学学报（医学版）, 2014, 43(3): 359-365.
[11] 徐倩，金梦媚，郑文文，朱丽，徐水凌. Toll样受体2/4-核因子κB信号通路在人结核分枝杆菌侵入小鼠树突细胞2.4中的作用[J]. 浙江大学学报（医学版）, 2014, 43(2): 200-206.
[12] 曾玲晖，丁美萍. mTOR信号通路在癫痫发生中的作用[J]. 浙江大学学报（医学版）, 2013, 42(6): 597-601.
[13] 刘欣，吴海琴. 促红细胞生成素的神经保护作用[J]. 浙江大学学报（医学版）, 2013, 42(6): 693-699.
[14] 张军, 张国新, 陈菲菲, 何邦顺, 叶峰, 潘晓林. 幽门螺杆菌脂多糖对胃粘膜shh信号通路相关蛋白Gli、Ptch-1表达的影响[J]. 浙江大学学报（医学版）, 2013, 42(5): 543-549.
[15] 饶翠, 林山力, 文欢, 邓红. 经典转化生长因子β/Smad信号和Wnt/β-catenin信号间的相互作用[J]. 浙江大学学报（医学版）, 2013, 42(5): 591-597.